Mutated Xylanase Gene with a Broad PH Range of Reaction and Site-Specific Mutagenesis Method Thereof

ABSTRACT

A mutated xylanase gene with a broad pH range of reaction includes a forty-first amino acid or a twenty-first amino acid generated from transforming asparagine to aspartic acid so as to form the mutated xylanase gene. A site-specific mutagenesis method includes the step of: mutating the forty-first amino acid or the twenty-first amino acid of the xylanase gene by transforming asparagine to aspartic acid so as to form the mutated xylanase gene.

BACKGROUND OF THE INVENTION

1. Field of the Invention

The present invention relates to a mutated xylanase gene with a broad pHrange of reaction and a site-specific mutagenesis method thereof. Moreparticularly, the present invention relates to the site-specificmutagenesis method utilized to mutate a forty-first amino acid or atwenty-first amino acid of a xylanase gene from asparagine to asparticacid so as to form the xylanase gene with the broad pH range ofreaction.

2. Description of the Related Art

Generally, most of xylans widely exist in structural polysaccharides ofplants. The xylan can naturally function as a protective material forcelluloses of plans such that the protective material can be alimitation in processing the natural material of plants. For example, inmanufacturing pulps of paper materials, there is a need of using achloride material as a bleaching agent to bleach the pulp due to thefact that the xylan and lignin adhere to surfaces of the celluloses ofthe plants. After processing the bleaching procedure, the reactedchloride may produce residual products of chemicals which are toxic andcarcinogenic substances. The toxic and carcinogenic substances arepersistent and bioaccumulating in the natural environment. Thisseriously destroys the natural environment and the ecological system.

In the livestock industry, animal feed is widely fed and delivered toanimal digestive system. The animal feed naturally contains cellulosesand hemicelluloses of plants with which to cover its valuable nutrients.The celluloses and hemicelluloses of plants separate the valuablenutrients from enzyme existing in the animal digestive system. In thismanner, the valuable nutrients of the animal feed cannot be reacted withthe enzyme, or cannot be absorbed by animal intestines of the digestivesystem. Accordingly, this affects the growth of animals. If theundigested nutrients are excreted from the animal digestive system,there are pollution sources of the undigested nutrients which causeenvironmental pollution. Hence, there is a need for removal of the xylanfrom the celluloses and hemicelluloses of plants.

Generally, there is a conventional xylanase which is separated from arumen microorganism and can be widely used to eliminate the aboveproblem due to the fact that the xylanase can decompose the xylan. Inthe papermaking industry, the xylanases can decompose the hemicellulosesexisting in the paper pulp such that links between the lignin and thecelluloses, and between the lignin and the hemicelluloses. Accordingly,the lignin can be released from the paper pulp in the bleaching process.In the food-processing industry, an oligosaccharide is used not only todiscompose the hemicelluloses in fruit juices, but also to be as rawmaterials of foods. In the livestock industry, the oligosaccharide isadded to the animal feed. In this manner, the xylanases of theoligosaccharide can be utilized to decompose the xylan in attempting toaid the valuable nutrients to be absorbed by animal intestines of thedigestive system. Accordingly, this results in an increase of theabsorbed mount of the valuable nutrients.

The primary problem occurring during use of the conventional xylanasesis due to the fact that the xylanases possess a lower degree ofdurability in the broad pH range. Disadvantageously, there is alimitation of use of the xylanases which cannot be widely used invarious industries. By way of example, in the papermaking industry,there is a further need of the xylanases for sustaining a higher degreeof base materials. Inevitably, the conventional xylanase separated fromthe rumen microorganism, with a poor durability in the broad pH range,is unsuitable for use in the papermaking industry.

It is a common practice that a mutation method is utilized to improve acharacteristic of enzyme in the art. A conventional mutation method isdisclosed in the book by Joshi et al. entitled “Hydrogen Bonding andCatalysis”: “a novel explanation for how a single amino acidsubstitution can change the pH optimum of a glycosidase,” J. Mol. Biol.(2000) 299, 255-279. A thirty-fifth amino acid of a xylanase gene ofbacillus circulans is mutated from asparagine to aspartic acid forreducing a pKa value of the bacillus circulans so as to enhance itsacid-resistibility. However, this conventional mutation method partiallyenhances the acid-resistibility of the xylanase gene, and cannoteffectively broaden the pH range of reaction of the xylanase gene.

As is described in greater detail below, the present invention intendsto provide a mutated xylanase gene with a broad pH range of reaction anda site-specific mutagenesis method thereof. The site-specificmutagenesis method is processed to mutate a forty-first amino acid or atwenty-first amino acid of a xylanase gene from asparagine to asparticacid so as to form the xylanase gene with the broad pH range of reactionin such a way as to mitigate and overcome the above problem.Advantageously, the mutated xylanase gene of the present invention issuccessful in enhancing its acid/base-resistibility, broadening a pHrange of reaction and increasing its reaction activity.

SUMMARY OF THE INVENTION

The primary objective of this invention is to provide a mutated xylanasegene with a broad pH range of reaction. The xylanase gene with the broadpH range of reaction is generated from mutating a forty-first amino acidor a twenty-first amino acid of a xylanase gene from asparagine toaspartic acid which does not reduce reaction activity of the xylanasegene.

The secondary objective of this invention is to provide a site-specificmutagenesis method for broadening the pH range of reaction of xylanases.The site-specific mutagenesis method is processed to mutate at least oneamino acid of an enzyme gene from asparagine to aspartic acid so as toform a mutated gene of the enzyme. Accordingly, the site-specificmutagenesis method is achieved in broadening the pH range of reaction,and enhancing its reaction activity of the enzyme.

The mutated xylanase gene in accordance with an aspect of the presentinvention includes a forty-first amino acid or a twenty-first amino acidof the xylanase gene being mutated by transforming asparagine toaspartic acid so as to form the mutated xylanase gene.

In a separate aspect of the present invention, the site-specificmutagenesis method includes the step of mutating the forty-first aminoacid or the twenty-first amino acid of the xylanase gene by transformingasparagine to aspartic acid so as to form the mutated xylanase gene.

Further scope of the applicability of the present invention will becomeapparent from the detailed description given hereinafter. However, itshould be understood that the detailed description and specificexamples, while indicating preferred embodiments of the invention, aregiven by way of illustration only, since various will become apparent tothose skilled in the art from this detailed description.

BRIEF DESCRIPTION OF THE DRAWINGS

The present invention will become more fully understood from thedetailed description given hereinbelow and the accompanying drawingswhich are given by way of illustration only, and thus are not limitativeof the present invention, and wherein:

FIG. 1 is a flow chart illustrating a site-specific mutagenesis methodfor a mutated xylanase gene with a broad pH range of reaction inaccordance with a preferred embodiment of the present invention;

FIG. 2 is a schematic view illustrating a genetic sequence (sequence IDnumber 3) of a mutated xylanase gene with a broad pH range of reactionin accordance with a first embodiment of the present invention;

FIG. 3A is a SDS-PAGE analysis image of a wild-type xylanase gene inaccordance with the first embodiment of the present invention;

FIG. 3B is a SDS-PAGE analysis image of a mutated-type xylanase inaccordance with the first embodiment of the present invention;

FIG. 4 is a chart illustrating relative enzyme activities of thewild-type xylanase and the mutated-type xylanase in accordance with thefirst embodiment of the present invention in relation to pH values;

FIG. 5 is a chart illustrating relative enzyme activities of thewild-type xylanase and the mutated-type xylanase in accordance with thefirst embodiment of the present invention in relation to time; and

FIG. 6 is a schematic view illustrating a genetic sequence (sequence IDnumber 4) of a mutated xylanase gene with a broad pH range of reactionin accordance with a second embodiment of the present invention.

DETAILED DESCRIPTION OF THE INVENTION

Turning now to FIG. 1, a flow chart of a site-specific mutagenesismethod for a mutated xylanase gene with a broad pH range of reaction inaccordance with the preferred embodiment of the present invention isillustrated. The site-specific mutagenesis method of the preferredembodiment of the present invention includes the steps of: utilizingcarriers to generate a plurality of xylanase genes which is designatedas step “S1”; executing a polymerase chain reaction which is designatedas step “S2”. In step “S1”, the carriers are utilized to generate thexylanase genes to form a plurality of first recombinant plasmids formass production of the xylanase genes; and the first recombinantplasmids are transformed into competent cells so as to form a pluralityof wild-type expression carriers. In step “S2”, the first recombinantplasmids are mixed with dNTP, reaction buffer, forward primer, reverseprimer and polymerase for processing the polymerase chain reaction so asto form second recombinant plasmids, and the second recombinant plasmidsare transformed into the competent cells so as to form a plurality ofmutated-type expression carriers. Since the polymerase chain reactioncan reproduce a great number of the xylanase genes and each of theforward primer and the reverse primer has a mutation position, thereproduction of the xylanase genes in the polymerase chain reaction cangenerate the mutated xylanase gene with a broad pH range of reaction. Inthis manner, a forty-first amino or a twenty-first amino acid of thexylanase gene is mutated from asparagine to aspartic acid by controllingthe forward primer and the reverse primer so as to form the mutatedxylanase gene with a broad pH range of reaction.

With continued reference to FIG. 1, the site-specific mutagenesis methodin accordance with the preferred embodiment of the present invention isimplemented by executing the first step “S1” of utilizing carriers togenerate xylanase genes. In step “S1”, the carriers are utilized togenerate the xylanase genes to form the first recombinant plasmids formass production of the xylanase genes; and the first recombinantplasmids are further transformed into the competent cells so as to formthe wild-type expression carriers. A genetic sequence of the wild-typexylanase gene used herein has been registered in a genetic sequencedatabase of GenBank database (accession number AY941119). The wild-typexylanase gene is separated from rumen microorganisms. A pET system forproducing the carriers used herein is shown for exemplification and notby way of limitation. The pET system is operated as follows:

The wild-type xylanase gene is preserved in a plasmid so as to form axylanase-gene-contained recombinant plasmid. Preferably, the plasmid isselected from pGEXSX-1 (Amersham Pharmacia, Sweden). The recombinantplasmids are transformed into first microorganisms which are inoculatedin a cultivation liquid containing antibiotics. In a preferredembodiment the first microorganism is selected from colon bacillus DH5 α(E. coli DH5α). In a preferred embodiment, the cultivation liquid isselected from Luria-Bertani broth cultivation liquid containingantibiotics. Preferably, the antibiotic is selected from ampicillinwhich has a concentration of 100 μg/mL. Next, the first microorganism iscultivated for 16 hours at 37 degrees Centigrade. Preferably, a plasmidpurification kit (commercially available from mini-M™ plasmid DNAextraction system, Viogene, Taiwan) is utilized to process and purifythe plasmids so as to generate purified recombinant plasmids.Subsequently, two restriction enzymes are utilized to cut the purifiedrecombinant plasmids. Preferably, the two restriction enzymes areselected from BamHI and NotI. After cutting the first plasmids, a DNAligase is utilized to react a DNA ligation for combining thexylanase-gene-contained DNA fragments with the broken first plasmids soas to form the first recombinant plasmids containing xylanase gene.Preferably, the first recombinant plasmids are selected from pET21C(Novagen, USA) and the DNA ligase is selected from a T4 ligase (Roche,Germany). In this circumstance, the operation of the pET system iscompleted. Subsequently, the first recombinant plasmids are transformedinto the competent cells which are confirmed by means of DNA sequencing.Preferably, the competent cells are selected from colon bacillus DH5α.Accordingly, the first step “S1” is completely executed.

With continued reference to FIG. 1, the site-specific mutagenesis methodin accordance with the preferred embodiment of the present invention isimplemented by executing the second step “S2” of executing a polymerasechain reaction. In step “S2”, the first recombinant plasmids are mixedwith dNTP, reaction buffer, forward primer, reverse primer andpolymerase for processing the polymerase chain reaction so as to formthe second recombinant plasmids, and the second recombinant plasmids arefurther transformed into the competent cells so as to form themutated-type expression carriers. In operation, the first recombinantplasmids, dNTP, reaction buffer, forward primer, reverse primer andpolymerase are added in a 200 μL thin-wall centrifuge tube. Preferably,the amount of the first recombinant plasmid is 50 ng. The dNTP consistsof dATP, dTTP, dCTP and dGTP each of which preferably has aconcentration of 360 μM. The reaction buffer is selected from 10×reaction buffer with an amount of 5 μL. The forward primer and reverseprimer have a concentration of 300 nM. In a preferred embodiment, theforward primer has a genetic sequence selected from (sequence IDnumber 1) while the reverse primer has a genetic sequence selected from(sequence ID number 2), as best shown in TABLE 1. In TABLE 1, positionsof the genetic sequences of the forward primer and reverse primer areunderlined indicating that a mutation position of the genetic sequence.The polymerase is selected from 0.7 μL (3.75 units) of Expand longtemplate DNA polymerase (Roche, Germany). Finally, distilled water isadded to a total amount of 50 μL. After shortly centrifugal operation,the polymerase is disposed in a Polymerase Chain Reaction (PCR) machinewhich is preferably selected from Applied Biosystems 2007 PCR system(USA).

TABLE 1 Genetic Sequence of Forward Primer and Reverse Primer forwardprimer 5′GGTGGTGGTCAAGACCAACATAAAGGTG3′ sequence ID number 1 reverseprimer 5′CACCTTTATGTTGGTCTTGACCACCACC3′ sequence ID number 2

Turning now to FIG. 2, a schematic view of a genetic sequence (sequenceID number 3) of a mutated xylanase gene with a broad pH range ofreaction in accordance with a first embodiment of the present inventionis illustrated. In the polymerase chain reaction, the xylanase gene isdenatured in high temperature. Next, the forward primer or the reverseprimer and the denatured single-strand xylanase gene are annealing suchthat the forward primer and the reverse primer correspondingly determinetwo predetermined points of the denatured xylanase gene between which toduplicate a DNA fragment. Subsequently, the polymerase can causeextensions of the forward primer and the reverse primer along thedenatured single-strand xylanase genes to form the duplicated DNAfragment. In operation, the PCR machine is set at temperature of 95degrees Centigrade for 3 minutes, 95 degrees Centigrade for 45 secondsfor denaturing, 55 degrees Centigrade for 1 minute for annealing, and 68degrees Centigrade for 9 minutes for extension which is a cycle forpolymerase chain reaction. The PCR machine is repeatedly executed thecycle 20 times. Subsequently, the PCR machine is set at temperature of55 degrees Centigrade for 1 minute, 68 degrees Centigrade for 15minutes, and is dropped to 4 degrees Centigrade so as to obtain reactionproducts of the polymerase chain reaction. Consequently, the polymerasechain reaction is completed. The second recombinant plasmids containingmutated xylanase gene with a broad pH range of reaction are formed bymeans of the polymerase chain reaction.

Next, a restriction enzyme is added to 20 μL of the reaction product ofthe polymerase chain reaction so as to cut the unmutated firstrecombinant plasmids in the reaction product of the polymerase chainreaction. Preferably, the restriction enzyme is selected from 1 μL ofDpnI reacting at temperature of 37 degrees Centigrade for 1 hour, 65degrees Centigrade for 10 minutes such that the second recombinantplasmids are transformed into the first microorganisms so as to form themutated-type expression carriers. The mutated-type expression carriersare cultivated and sieved in the antibiotic-contained cultivationliquid. Finally, three transformed colonies are selected and the secondrecombinant plasmids are confirmed by means of sequencing. Accordingly,the first step “S2” is completely executed. Since each of the forwardprimer and the reverse primer has a mutation position, the reproductionof the xylanase genes in the polymerase chain reaction can generate thesecond recombinant plasmids containing the mutated xylanase gene with abroad pH range of reaction. In this manner, the forty-first amino acidof the xylanase gene is mutated from asparagine to aspartic acid so asto form the mutated xylanase gene with a broad pH range of reaction. Themutated xylanse gene has the genetic sequence (sequence ID number 3)shown in FIG. 2. In FIG. 2, the forty-first amino acid of the mutatedxylanase gene as well as the aspartic acid is indicated in a frame.

The difference between the mutated xylanase gene with a broad pH rangeof reaction in accordance with the present invention and the xylanasegene are verified. Each of the mutated xylanase gene with a broad pHrange of reaction in accordance with the present invention and thexylanase gene is utilized to produce a wild-type xylanase gene and amutated-type xylanase gene for use in measuring reaction activity, a pHrange of reaction and base-resistibility of the enzyme. In comparisonwith the wild-type xylanase gene, the mutated xylanase gene inaccordance with the present invention can enhance the reaction activityand base-resistibility of the enzyme, and increase the pH range ofreaction.

Turning now to FIG. 3A, a SDS-PAGE (sodium dodecylsulfate-polyacrylamide gel electrophotesis) analysis image of awild-type xylanase gene in accordance with the first embodiment of thepresent invention is illustrated. Turning to FIG. 33B, a SDS-PAGEanalysis image of a mutated-type xylanase gene in accordance with thefirst embodiment of the present invention is illustrated. Firstly, thefirst recombinant plasmids are extracted from the wild-type expressioncarriers, and are transformed into second microorganisms so as to formgrowth carriers that contain the xylanase gene. Preferably, the secondmicroorganism is selected from colon bacillus BL21 (DE3). The growthcarriers are inoculated in 5 mL of an antibiotic-contained cultivationliquid to produce a bacteria liquid which is cultivated for 16 hours at37 degrees Centigrade and is vibrated at 255 rpm by a shaker. Aftercompletely cultivating the cultivation liquid, 5 mL of the bacterialiquid is further inoculated in 500 mL of the antibiotic-containedcultivation liquid which is cultivated at 37 degrees Centigrade and isvibrated at 180 rpm by a shaker. When a value of OD₆₀₀ of the bacterialiquid is 0.6-0.8, a medium of IPTC (isopropyl-β-D-thiogalactoside) isadded as a revulsive. Preferably, the IPTG has a final concentration of1 mM. The wild-type xylanase is generated after the revulsion of IPTGfor 4 hours. Subsequently, 4,000 g of the bacteria liquid is processedfor 20 minutes to precipitate bacteria by a centrifuge. The bacteria aredissolved in a citric acid buffer. Preferably, the citric acid bufferhas a pH value of 6 and a concentration of 50 mM. Subsequently,phenylmethylsulfonyl fluoride (PMSF) and leupeptin are added in thebacteria liquid as a protease inhibitor so as to avoid the protease inthe second microorganisms dissolving the wild-type xylanase. Preferably,the PMSF has a final concentration of 0.5 mM and the leupeptin has afinal concentration of 1 μg/mL. The bacteria are broken by ultrasonic toobtain a crude enzyme liquid. 10,000 g of the crude enzyme liquid isprocessed and separated for 30 minutes by a centrifuge. Furthermore, thecrude enzyme liquid is purified in a CM-Sepharose column and Ni-NTAaffinity column for purification so as to obtain the purified wild-typexylanase. Finally, the purified wild-type xylanase is dialyzed to removeredundant salts and to replace the citric acid buffer. Accordingly, thepurified wild-type xylanase is prepared and can be applied in thefollowing measuring procedure.

A manufacturing method for the mutated-type xylanase is identical withthat for the wild-type xylanase which is incorporated herein byreference. The extraction of the first recombinant plasmids from thewild-type expression carriers is only changed to the extraction of thesecond recombinant plasmids from the mutated-type xylanase. However, thedetailed descriptions for the extractions of the second recombinantplasmids from the mutated-type xylanase are omitted for the sake ofsimplicity. Accordingly, the mutated-type xylanase is prepared and canbe applied in the following measuring procedure.

With continued reference to FIGS. 3A and 3B, the wild-type xylanase andthe mutated-type xylanase are further analyzed by SDS-PAGE to identifytheir purification statuses and molecular weight. In FIGS. 3A and 3B,columns 1 a and 1 b represent a mark of molecular weight for standardprotein; column 2 a represents a crude enzyme liquid formed from thefirst recombinant plasmids; column 3 a represents the wild-type xylanasepurified in the CM-Sepharose column; column 4 a represents the wild-typexylanase purified in the Ni-NTA affinity column; column 2 b represents acrude enzyme liquid formed from the first recombinant plasmids; column 3b represents the mutated-type xylanase purified in the CM-Sepharosecolumn; column 4 b represents the mutated-type xylanase purified in theNi-NTA affinity column. As indicated in FIGS. 3A and 3B, the molecularweights of the wild-type xylanase and the mutated-type xylanase areapproximately 34 KDa.

In TABLE 2, enzyme activities of the wild-type xylanase and themutated-type xylanase are measured in various purification stages, andare compared. Firstly, 5 ng of the wild-type xylanase is added to asubstrate. Preferably, the substrate is selected from a liquid buffercontaining 20 mg/mL of soluable oat spelt xylan. The liquid buffer isselected from 50 mM of citric acid buffer which has a pH value of 6.5.After mixed, the wild-type xylanase buffer is reacted at the temperatureof 50 degrees Centigrade for 10 minutes such that the xylanase candecompose the xylan contained in the substrate. Subsequently, a methodof DNS (dinitrosalicylic acid) is utilized to process quantitativereduction for the redundant of the xylan remained in the substrate so asto obtain indexes of enzyme activities (U/mg). A unit activity (U) isthe substrate activity of catalyzing 1 μmole per minute. Preferably, aBCA protein quantitative set (available from Pierce Ltd., USA) can beutilized to quantitate the concentration of the wild-type xylanase.

A measuring method for the activity of mutated-type xylanase isidentical with that for the wild-type xylanase which is incorporatedherein by reference. Hence, the detailed descriptions for the measuringmethod for the activity of mutated-type xylanase are omitted for thesake of simplicity. The enzyme activity of mutated-type xylanase isslightly greater than that of the wild-type xylanase, as indicated inTABLE 2. Advantageously, the xylanase gene with the broad pH range ofreaction in accordance with the present invention does not decrease itsenzyme activity.

TABLE 2 Enzyme Aactivities of Wild-Type Xylanase and Mutated-TypeXylanase enzyme activity enzyme activity of wild-type of mutated-purification stage xylanase (U/mg) type xylanase Crude enzyme liquid2568.27 2124.9 CM-Sepharose column 14568.65 17446.2 Ni-NTA affinitycolumn 23244.85 25784.9

Turning now to FIG. 4, a chart illustrating relative enzyme activitiesof the wild-type xylanase and the mutated-type xylanase in relation topH values is shown. In order to demonstrate the relative enzymeactivities of the wild-type xylanase and the mutated-type xylanase invarious pH values, an enzyme pH optimal reaction test for the wild-typexylanase and the mutated-type xylanase is processed. 5 ng of thewild-type xylanase is added to 295 μg of substrate which is selectedfrom a liquid buffer containing 20 mg/mL of soluable oat spelt xylan. Inan example, the liquid buffer is selected from glycine buffer which hasa range of pH value from 2.0 to 3.5. In another example, the liquidbuffer is selected from citric acid buffer which has a range of pH valuefrom 3.0 to 6.5. In another example, the liquid buffer is selected fromphosphate buffer which has a range of pH value from 6.0 to 7.5. Inanother example, the liquid buffer is selected from Tris buffer whichhas a range of pH value from 7.0 to 10.0. In another example, the liquidbuffer is selected from CAPS buffer which has a range of pH value from10.0 to 11.0. After mixed, the wild-type xylanase buffer is reacted atan appropriate temperature for 10 minutes such that the xylanase candecompose the xylan contained in the substrate. Subsequently, the methodof DNS (dinitrosalicylic acid) is utilized to process quantitativereduction for the redundant of the xylan remained in the substrate so asto estimate indexes of enzyme activities. The enzyme pH optimal reactiontest is operated at a pH standard of 6.5 so as to further estimate otherpH values of the enzyme activities.

With continued reference to FIG. 4, the operation for measuring theactivity of mutated-type xylanase is identical with that for thewild-type xylanase which is incorporated herein by reference. Hence, thedetailed descriptions for the operation for measuring the activity ofmutated-type xylanase are omitted for the sake of simplicity. In FIG. 4,when the mutated-type xylanase is reacted at pH 2, the mutated-typexylanase has a 60 percent activity with respect to the activity of thepH optimal value. In this condition, the wild-type xylanase has a 20percent activity with respect to the activity of the pH optimal value.When the wild-type and mutated-type xylanases are reacted at pH 11, themutated-type xylanase has a 90 percent activity with respect to theactivity of the pH optimal value while the wild-type xylanase has an 80percent activity. However, it appears that the mutated-type xylanase inaccordance with the present invention has a greater reaction activity inthe acid or base environment than that of the wild-type xylanase.Accordingly, the mutated-type xylanase in accordance with the presentinvention has a broad pH range of reaction.

Turning now to FIG. 5, a chart illustrating relative enzyme activitiesof the wild-type xylanase and the mutated-type xylanase in relation totime (hour) is shown. The mutated-type xylanase is placed in a baseenvironment with pH 11 at the room temperature. Subsequently, each of 5ng of the mutated-type xylanase is obtained in a series of time, and isadded to 295 μg of substrate. After mixed, the substrate is placed at anappropriate temperature and a pH value for 10 minutes. Subsequently, themethod of DNS (dinitrosalicylic acid) is utilized to processquantitative reduction for the redundant of the xylan remained in thesubstrate so as to estimate indexes of enzyme activities.

In FIG. 5, when the substrate is placed for 30 minutes, the mutated-typexylanase has an 80 percent activity with respect to the activity of thepH optimal value. When the mutated-type xylanase is placed for 4 hours,the mutated-type xylanase has a 70 percent activity with respect to theactivity of the pH optimal value. When the mutated-type xylanase isplaced for 6 to 24 hours, the mutated-type xylanase still has a 55percent activity with respect to the activity of the pH optimal value.Advantageously, it appears that the mutated-type xylanase and thesite-specific mutagenesis method thereof in accordance with the firstembodiment of the present invention are successful in enhancing itsacid/base-resistibility.

Turning now to FIG. 6, a schematic view of a genetic sequence (sequenceID number 4) of a mutated xylanase gene with a broad pH range ofreaction in accordance with a second embodiment of the present inventionis illustrated. The mutated xylanse gene has the genetic sequence(sequence ID number 4) shown in FIG. 6. In FIG. 6, a mutation positionof the twenty-first amino of the mutated xylanase gene as well as theaspartic acid is indicated in a frame. The sequence ID number 4 is agenetic sequence from 21^(st) to 100 amino of the sequence ID number 3.Although the mutation positions of the sequence ID numbers: 3 and 4 aredifferent, the amino positions of the sequence ID numbers: 3 and 4 aresubstantially the same. Advantageously, it appears that the mutated-typexylanase and the site-specific mutagenesis method thereof in accordancewith the second embodiment of the present invention are successful inenhancing its acid/base-resistibility, and enhancing its reactionactivity of the enzyme.

As has been previously described, the site-specific mutagenesis methodin accordance with the present invention is utilized to mutate aforty-first amino acid or a twenty-first amino acid of an enzyme genefrom asparagine to aspartic acid so as to form the enzyme gene with thebroad pH range of reaction and to enhance its base-resistibility.Preferably, the enzyme is selected from oxidoreductases, transferase,hydrolase, lipase, isomerase or is synthase. Preferably, the hydrolaseis selected from the xylanase.

In addition to this, the mutated xylanase gene with a broad pH range ofreaction in accordance with the present invention can be furtherutilized and incorporated into a plasmid or a chromosome by means of therecombinant DNA technology. In another embodiment, the mutated xylanasegene in accordance with the present invention can be incorporated into acell by means of a genetic engineering process.

As has been discussed above, the conventional xylanases possess a lowerdegree of durability in the broad pH range such that the xylanasescannot be widely used in various industries. Conversely, thesite-specific mutagenesis method in accordance with the presentinvention is processed to mutate at least one amino acid of the xylanasegene from asparagine to aspartic acid so as to form the mutated xylanasegene with a broad pH range of reaction. Advantageously, the mutatedxylanase gene and the site-specific mutagenesis method in accordancewith the present invention are successful in broadening the pH range ofreaction of the xylanase and enhancing activities of reaction of thexylanase.

Although the invention has been described in detail with reference toits presently preferred embodiment, it will be understood by one ofordinary skill in the art that various modifications can be made withoutdeparting from the spirit and the scope of the invention, as set forthin the appended claims.

1. A mutated xylanase gene with a broad pH range of reaction, comprising: a forty-first amino acid of a xylanase gene being mutated to aspartic acid.
 2. The mutated xylanase gene with a broad pH range of reaction as defined in claim 1, wherein the mutated xylanase gene has a genetic sequence of sequence ID number
 3. 3. The mutated xylanase gene with a broad pH range of reaction as defined in claim 1, wherein carriers are utilized to generate the xylanase genes to form a plurality of first recombinant plasmids for mass production of the xylanase genes.
 4. The mutated xylanase gene with a broad pH range of reaction as defined in claim 3, wherein a pET system is used to produce the carriers.
 5. The mutated xylanase gene with a broad pH range of reaction as defined in claim 3, wherein the first recombinant plasmids are mixed with dNTP, reaction buffer, forward primer, reverse primer and polymerase for processing a polymerase chain reaction so as to form second recombinant plasmids.
 6. The mutated xylanase gene with a broad pH range of reaction as defined in claim 5, wherein the forward primer has a genetic sequence selected from sequence ID number 1 and the reverse primer has a genetic sequence selected from sequence ID number 2; and wherein the sequence ID number 1 is 5′GGTGGTGGTCAAGACCAACATAAAGGTG3′ and the sequence ID number 2 is 5′CACCTTTATGTTGGTCTTGACCACCACCACC3′.
 7. The mutated xylanase gene with a broad pH range of reaction as defined in claim 1, wherein the mutated xylanase gene is incorporated into a plasmid or a chromosome by means of a recombinant DNA technology.
 8. The mutated xylanase gene with a broad pH range of reaction as defined in claim 1, wherein the xylanase gene is separated from rumen microorganism which has accession No. AY941119 registered in a genetic sequence database of GenBank database.
 9. A mutated xylanase gene with a broad pH range of reaction, comprising: a twenty-first amino acid of a xylanase gene being mutated to aspartic acid.
 10. The mutated xylanase gene with a broad pH range of reaction as defined in claim 9, wherein the mutated xylanase gene has a genetic sequence of sequence ID number
 4. 11. The mutated xylanase gene with a broad pH range of reaction as defined in claim 9, wherein carriers are utilized to generate the xylanase genes to form a plurality of first recombinant plasmids for mass production of the xylanase genes.
 12. The mutated xylanase gene with a broad pH range of reaction as defined in claim 11, wherein a pET system is used to produce the carriers.
 13. The mutated xylanase gene with a broad pH range of reaction as defined in claim 11, wherein the first recombinant plasmids are mixed with dNTP, reaction buffer, forward primer, reverse primer and polymerase for processing a polymerase chain reaction so as to form second recombinant plasmids.
 14. The mutated xylanase gene with a broad pH range of reaction as defined in claim 13, wherein the forward primer has a genetic sequence selected from sequence ID number 1 and the reverse primer has a genetic sequence selected from sequence ID number 2; and wherein the sequence ID number 1 is 5′GGTGGTGGTCAAGGACCAACATAAAGGTG3′ and the sequence ID number 2 is 5′CACCTTTATGTTGGTCTTGACCACCACC3′.
 15. The mutated xylanase gene with a broad pH range of reaction as defined in claim 9, wherein the mutated xylanase gene is incorporated into a plasmid or a chromosome by means of a recombinant DNA technology.
 16. The mutated xylanase gene with a broad pH range of reaction as defined in claim 9, wherein the xylanase gene is separated from rumen microorganism which has accession No. AY941119 registered in a genetic sequence database of GenBank database.
 17. A site-specific mutagenesis method for a mutated enzyme gene with a broad pH range of reaction, comprising the step of: mutating at least one amino acid of an enzyme gene by transforming asparagine to aspartic acid so as to form the mutated enzyme gene.
 18. The site-specific mutagenesis method for a mutated enzyme gene with a broad pH range of reaction as defined in claim 17, wherein the enzyme gene is selected from a xylanase gene, and the amino acid of the enzyme gene is a forty-first amino acid or a twenty-first amino of the xylanase gene.
 19. The site-specific mutagenesis method for a mutated enzyme gene with a broad pH range of reaction as defined in claim 18, wherein the mutated xylanase gene has a genetic sequence of sequence ID number 3 while the amino acid of the enzyme gene is the forty-first amino acid of the xylanase gene.
 20. The site-specific mutagenesis method for a mutated enzyme gene with a broad pH range of reaction as defined in claim 18, wherein the mutated xylanase gene has a genetic sequence of sequence ID number 4 while the amino acid of the enzyme gene is the twenty-first amino acid of the xylanase gene. 